Reverse Complement Adapters for NGS UMI Hopping

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Solution Overview

Problem

Next-generation sequencing (NGS) technologies face challenges in differentiating between true rare variants and errors introduced during sample preparation, particularly due to 'UMI hopping' where fragments acquire multiple UMI tags during PCR, leading to incorrect identification of unique sequences and overestimation of library complexity.

Innovation Solution

The use of novel adapter structures with a UMI on the 5' P5 adapter instead of the 3' P7 adapter, employing reverse complement adapters to maintain the standard protocol while reducing UMI hopping, thereby ensuring accurate conversion of DNA samples into sequencing libraries with minimized errors.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of manufacture

If UMI is placed on the 3' P7 adapter, then library construction follows standard protocol, but UMI hopping occurs during PCR leading to erroneous identification of unique sequences

Engineering Contradiction:

Improvelibrary constructionVSAvoidsequence identification

Core Design Contradiction:

Ease of manufactureVSReliability

Solution Approach 1:

The patent inverts the conventional adapter design by placing the UMI on the 5' P5 adapter instead of the 3' P7 adapter. This reversal prevents the UMI from being copied during PCR amplification, as the P5 adapter does not serve as a PCR template, thereby eliminating UMI hopping while maintaining standard library construction protocols

Inventive Principle:

Principle #13The other way round (Inversion)

2Device complexity

If standard P5 and P7 adapters with UMI on P7 are used, then protocol is simple, but fragments acquire multiple UMIs during PCR amplification

Engineering Contradiction:

Improveadapter structureVSAvoidUMI tagging accuracy

Core Design Contradiction:

Device complexityVSManufacturing precision

Solution Approach 1:

The patent extracts the UMI from the P7 adapter and relocates it to the P5 adapter. This separation removes the problematic interaction between the UMI-containing P7 adapter and the PCR amplification process, preventing the erroneous acquisition of multiple UMIs while maintaining adapter structure simplicity

Inventive Principle:

Principle #2Taking out (Extraction)

3Quantity of substance

If UMI hopping occurs during PCR, then library complexity is overestimated, but true rare variants cannot be differentiated from errors

Engineering Contradiction:

Improvelibrary complexityVSAvoidvariant detection

Core Design Contradiction:

Quantity of substanceVSMeasurement precision

Solution Approach 1:

The patent applies preliminary anti-action by positioning the UMI on the P5 adapter before PCR amplification begins. This preemptive measure ensures that the UMI will not be copied during PCR, preventing the generation of erroneous duplicate sequences with different UMIs and enabling accurate differentiation between true rare variants and PCR errors

Inventive Principle:

Principle #9Preliminary anti-action

Data Source

PatentUS11053537B2Reverse complement adapters for the mitigation of UMI hopping

Publication Date: 2021.07.06 INTEGRATED DNA TECHNOLOGIES INC

AI summary

The invention pertains to construction of next-generation DNA sequencing (NGS) libraries for whole genome sequencing, targeted resequencing, sequencing-based screening assays, metagenomics, or any other application requiring sample preparation for NGS.